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|Title: ||Negative Regulatory Mechanisms Underlying EPO Receptor Signaling and Erythropoiesis|
|Authors: ||Richmond, Terri|
|Advisor: ||Barber, Dwayne|
|Department: ||Medical Biophysics|
|Issue Date: ||2-Mar-2010|
|Abstract: ||Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis. Fundamental to this action is the ability of EPO to bind the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Positive regulation of intracellular signal transduction pathways downstream of EPO-R and JAK2 are well documented, but less is known about negative regulation of EPO-R signaling. Two distinct experimental strategies were utilized to examine a subset of the negative regulatory mechanisms underlying EPO-R signaling and erythropoiesis.
Mice deficient in the E3 ubiquitin ligase, Cbl, were generated previously and displayed elevated platelet numbers, expansion of splenic red pulp and splenomegaly, suggesting that Cbl-/- mice have defects in megakaryocyte/erythrocyte progenitors or more committed cells of each lineage. Our studies illustrated that genetic ablation of Cbl resulted in elevated total numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid, but decreased bone marrow-derived late erythroblasts. Cbl-deficient late erythroblasts displayed elevated apoptosis, as well as increased expression of Foxo3a and increased mRNA levels of the pro-apoptotic genes, Bim and FasL. These studies implicate Cbl as an important negative regulator of multiple facets of erythroid signaling.
The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome led us to examine the role of EPO-R ubiquitination on signal transduction and proliferation. Lysine mutagenesis of EPO-R showed that K348, K388 and K428 were the primary ubiquitin acceptor sites when EPO-R mutants were expressed in HEK 293T cells. BaF3 cells expressing an EPO-R deficient in cytoplasmic lysines displayed diminished EPO-mediated EPO-R, JAK2, PKB and STAT5 phosphorylation and could not proliferate in response to EPO. The membrane proximal lysines of EPO-R, K256 and K276, were necessary for proliferation at physiologic EPO concentrations but were not required at saturating EPO concentrations. Single lysine EPO-R add-back mutants restored signaling and proliferation to BaF3 cells at physiologically elevated EPO concentrations, signifying that EPO-R lysines finely mediate EPO-dependent proliferation and signal transduction. These analyses demonstrate a positive regulatory role for lysines in signal transduction and proliferation.|
|Appears in Collections:||Doctoral|
Department of Medical Biophysics - Doctoral theses
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